WO2000042210A2 - Marqueurs d'adn microsatellites et leurs utilisations - Google Patents

Marqueurs d'adn microsatellites et leurs utilisations Download PDF

Info

Publication number
WO2000042210A2
WO2000042210A2 PCT/US2000/000325 US0000325W WO0042210A2 WO 2000042210 A2 WO2000042210 A2 WO 2000042210A2 US 0000325 W US0000325 W US 0000325W WO 0042210 A2 WO0042210 A2 WO 0042210A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
ssr
polynucleotide
locus
motif
Prior art date
Application number
PCT/US2000/000325
Other languages
English (en)
Other versions
WO2000042210A3 (fr
Inventor
Craig S. Echt
C. Dana Nelson
Original Assignee
International Paper Company
United States Of America, As Represented By The Secretary Of Agriculture
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/232,785 external-priority patent/US6733965B2/en
Application filed by International Paper Company, United States Of America, As Represented By The Secretary Of Agriculture filed Critical International Paper Company
Priority to AU24076/00A priority Critical patent/AU2407600A/en
Priority to NZ512941A priority patent/NZ512941A/en
Publication of WO2000042210A2 publication Critical patent/WO2000042210A2/fr
Publication of WO2000042210A3 publication Critical patent/WO2000042210A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to identification and isolation of the simple sequence repeat (SSR) loci in the higher eukaryotes, such as the plants, and particularly the pines.
  • SSR simple sequence repeat
  • SSR loci of the invention are particularly useful as genetic markers for genetic mapping, population genetics studies and inhe ⁇ tance studies in various plant breeding programs
  • Loblolly pine (Pinus taeda L.) is an important, expe ⁇ mental and commercial forest tree species native to the southeastern United States. Loblolly pine is planted extensively in the southeastern United States and to lesser degrees in other warm temperate regions of the world. In the United States, plantations are managed and utilized for a variety of products including raw mate ⁇ als (wood, fiber, and chemicals), ecosystem components (wildlife habitat and water and soil conservation), and recreational activities
  • Loblolly pine breeding has various limitations, such as, long generation times to flower (>5 years) and harvest (>15 years), low tolerance to inbreeding, large size of individual trees, variable sites for testing and replanting, difficulty of vegetative propagation, low heritability of important traits, and uncertainty of trait values.
  • MAS Marker-assisted selection
  • Important improvements afforded by MAS include reducing the time-to-selection and improving the accuracy of selection.
  • An important goal of such research is to identify DNA markers or other measures that predict performance of mature trees. With this information, tree breeders could more confidently select trees at an early age, induce them to flower, and breed them to produce the next generation.
  • selections made at an early age could be vegetatively propagated in mass using rooted cutting or tissue culture based technologies (Bradshaw and Foster 1992 Can. J. For. Res., 22:1044-1049.). Vegetative propagation and deployment has the potential to greatly improve wood and fiber yield and quality by capturing within-family genetic variation and providing better performing varieites for plantation establishment.
  • family-specific mapping and MAS approaches have potential, these methods are limited to situations where small breeding ( ⁇ 10 parents) populations are maintained with progeny established in large-family (n>500) tests. In practice, however, most loblolly pine breeding programs do not fit this situation. More typical is large breeding populations, sometimes several populations per program, and always relatively small-family (n ⁇ 150) progeny tests. In addition most programs now include many pedigrees of at least three-generations, with nearly mature third-generation trees in the field. Utilizing existing extensive pedigree and progeny test information is essential for developing better MAS technology and improving breeding programs.
  • SSR simple sequence repeat
  • SSRs Simple sequence repeats
  • microsatellite DNA repeats have now been discovered in the pines and have been shown to exhibit length polymorphisms. These repeats represent an abundant pool of potential genetic markers. Accordingly, in one aspect, the present invention relates to the plant SSR motifs, such as for example, di-, tri- and tetra-nucleotide repeated motifs.
  • the invention relates to the polynucleotides containing one or more such SSR motifs and the primers for the amplification of the fragments containing
  • the primers may be cloned polynucleotide fragments or chemically synthesized oligonucleotides, and contain at least a portion of the non-repeated, non-polymorphic sequence flanking SSRs on either 5' or 3' end.
  • the present invention is also directed to a kit for the rapid analysis of one or more specific DNA polymorphisms of the type described in this application
  • the kit includes oligodeoxynucleotide primers for the amplification of fragments containing one or more SSR sequences.
  • the invention provides for a method of analyzing one or more specific SSR polymorphisms in an individual or a population, which involves amplification of small segment(s) of DNA containing the SSR and non-repeated flanking
  • the invention provides for a method of determining the sequence information necessary for primer production by isolation and sequencing of DNA fragments containing the SSRs, using hybridization of a synthetic, cloned, amplified or genomic probe, containing sequences substantially homologous to the SSR, to the DNA.
  • the present invention is directed to a method for detecting the presence of a specific trait in a subject, such as a plant.
  • the method includes isolating the genomic DNA from the subject individual and analyzing the genomic DNA with a polymo ⁇ hic amplified DNA marker containing one or more SSR sequences.
  • the SSR markers of the invention are used in commercial plant breeding. Traits of economic importance in plant crops can be identified through linkage analysis using polymo ⁇ hic DNA markers.
  • SSR stands for a "simple sequence repeat” and refers to any short sequence, foi example, a mono-, di-, tri-, or tetra-nucleotide that is repeated at least once in a particular nucleotide sequence. These sequences are also known in the art as “microsatellites.”
  • a SSR can be represented by the general formula (N,N 2 ...N j ) n , wherein
  • N represents nucleotides A, T, C or G
  • i represents the number of the nucleotides in the base repeat
  • n represents the number of times the base is repeated in a particular DNA sequence.
  • the base repeat be., N,N 2 ...Ni, is also referred to herein as an "SSR motif.”
  • (ATC) 4 refers to a tri-nucleotide ATC motif that is repeated four times in a particular sequence.
  • (ATC) 4 is a shorthand version of
  • complement of a SSR motif refers to a complementary strand of the represented motif.
  • complement of (ATG) motif is (TAC).
  • permutations of a SSR motif refers to all possible combinations of a motif found within the repeated sequence of that motif.
  • permutations of the (ATG) 5 motif i.e., ATGATGATGATGATG
  • GAT as both can be found in this repeat.
  • perfect repeat refers to a repeated SSR motif without interruption and without adjacent repeat(s) of a different motif.
  • the repeats may be "imperfect” when a repeated SSR motif is interrupted by a number of non-repeated nucleotides, such as for example in (AC) 5 GCTAGT(AC) 7
  • AC GCTAGT(AC) 7
  • Other possible variations of SSRs would be known to those of skill in the art.
  • These repeats, including compound repeats, are defined by Weber, J.L., 1990, Genomics, 7:524-530.
  • compound repeat refers to a SSR that contains at least two different repeated motifs that may be separated by a stretch of non-repeated nucleotides.
  • An example of a compound repeat is (ATC) 5 (AT) 6 .
  • SSR locus refers to a location on a chromosome of a SSR motif; locus may be occupied by any one of the alleles of the repeated motif.
  • Allele is one of several alternative forms of the SSR motif occupying a given locus on the chromosome.
  • the (ATC) n locus refers to the fragment of the chromosome containing this repeat, while (ATC) 4 and (ATC) 7 repeats represent two different alleles of the (ATC) n locus.
  • locus refers to the repeated SSR motif and the flanking 5' and 3' non-repeated sequences. SSR loci of the invention are useful as genetic markers, such as for determination of polymo ⁇ hysm.
  • Polymo ⁇ hism is a condition in DNA in which the most frequent variant (or allele) has a population frequency which does not exceed 99%.
  • the term “heterozygosity” (H) is used when a fraction of individuals in a population have different alleles at a particular locus (as opposed to two copies of the same allele). Heterozygosity is the probability that an individual in the population is heterozygous at the locus. Heterozygosity is usually expressed as a percentage (%), ranging from 0 to 100%, or on a scale from 0 to 1.
  • the term “informativeness” is a measure of the utility of the polymo ⁇ hism. In general, higher informativeness means greater utility.
  • Informativeness is usually defined in terms of , either heterozygosity or "Polymo ⁇ hism Information Content” (PIC) (for PIC see Botstein, D., et al., 1980, Am. J. Hum. Genet., 32, 314-331).
  • PIC represents the probability that the parental origin of an allele can be determined from the marker genotype of the locus in any given offspring.
  • the PIC values range from 0 to 1.0, and are smaller in value than heterozygosities.
  • the formulas for calculating H and PIC are disclosed in the examples. For markers that are highly informative (heterozygosities exceeding about 70%), the difference between heterozygosity and PIC is slight.
  • Primers are short polynucleotides or oligonucleotides required for a polymerase chain reaction that are complementary to a portion of the polynucleotide to be amplified.
  • the phrase "primer adapted for detection of a SSR marker” means that the primer is capable of amplyfying a particular SSR locus to be used as a marker, wherein the primer is complementary to either the 5' or the 3' non-repeated region of the SSR locus and is of a length suitable for use as a primer.
  • the primer is no more than 50 nucleotides long, preferably less than about 30 nucleotides long, and most preferably less than about 24 nucleotides long.
  • polynucleotide is intended to include double or single stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and anti-sense strands together or individually (although only sense or anti- sense stand may be represented herein).
  • PNA protein nucleic acids
  • This also includes nucleic acids containing modified bases, for example thio-uracil, thio-guanine and fluoro-uracil.
  • nucleic acid or polynucleotide refers to a component that is removed from its original environment (for example, its natural environment if it is naturally occurring).
  • An isolated nucleic acid or polypeptide may contains less than about 50%, preferably less than about 75%, and most preferably less than about 90%, of the cellular components with which it was originally associated.
  • a polynucleotide amplified using PCR so that it is sufficiently and easily distinguishable (on a gel from example) from the rest of the cellular components is considered “isolated”.
  • the polynucleotides of the invention may be "substantially pure," i.e., having the highest degree of purity that can be achieved using purification techniques known in the art.
  • hybridization refers to a process in which a strand of nucleic acid joins with a complementary strand through base pairing.
  • Polynucleotides are "hybridizable" to eacli other when at least one strand of one polynucleotide can anneal to a strand of another polynucleotide under defined stringency conditions.
  • Hybridization requires that the two polynucleotides contain substantially complementary sequences; depending on the stringency of hybridization, however, mismatches may be tolerated.
  • hybridization of two sequences at high stringency (such as, for example, in an aqueous solution of 0.5X SSC at 65°C) requires that the sequences exhibit some high degree of complementarily over their entire sequence.
  • intermediate stringency such as, for example, an aqueous solution of 2X SSC at 65 °C
  • low stringency such as, for example, an aqueous solution of 2X SSC at 55 °C
  • IX SSC 0.15 M NaCl, 0.015 M Na citrate.
  • the above solutions and temperatures refer to the probe- washing stage of the hybridization procedure.
  • the term "a polynucleotide that hybridizes under stringent (low, intermediate) conditions" is intended to encompass both single and double-stranded polynucleotides although only one strand will hybridize to the complementary strand of another polynucleotide.
  • % identity refers to the percentage of the nucleotides of one polynucleotide that are identical to the nucleotides of another sequence of identical length (excluding the length of the SSR) as implemented by the National Center for
  • the % identity value may be determined using a PowerBlast program (Zhang and Madden 1977 Genome Res. 7:649-56.).
  • % homology between the sequences is a function of the number of matching positions shared by two sequences divided by the number of positions compared and then multiplied by 100. This comparison is made when two sequences are aligned (by introducing gaps if needed) to give maximum homology.
  • PowerBlast program implemented by the National Center for Biotechnology Information, is used to compute optimal, gapped alignments.
  • the % homology comparison may be determined using a Blast 2.0 program implemented by the National Center for Biotechnology Information.
  • the present invention relates to SSR motifs and SSR loci useful as genetic markers in various organisms, particularly plants.
  • the SSR motifs and loci originate from the pines, such as the pines of the Pinus genus, for example P. taeda, P. caribaea, P. ponderosa, P. radiata, P. resinosa, P. strobus, and P. sylvestris.
  • the pines and SSRs thereof of the present invention can belong to either of the two subgenera of the Pinus genus. P.
  • strobus (white pine) is a species of the Strobus subgenus
  • P. taeda P. caribaea
  • P. ponderosa P. radiata
  • P. resinosa P. sylvestris
  • SSR motifs of the invention have the general formula (N,, N 2 ...N,) n , wherein:
  • the total number of nucleotides in a motif (i) is about six, preferably four, three or two.
  • SSR motif of the above formula is within the scope of the invention, however, the following SSR motif are preferred: AC, AAC, AAG, AAT, ACC, ACG, AGG, ATC, AAAC, AAAT, AGAT and all complements and permutation of said motifs, such as for example ATG, CAT, TTG, TTA, TTC, ATT, and TAT.
  • Compound repeats are also within the scope of the invention. Examples of such repeats are: (A) n ...(ATG) n ; (ATG) n ...(C) n ; (CAT) n ...(A) n ; (ACC) n ...(ATC) n ; (TTG) n ...(TTA) n ; (C.) n ...(ATT)minded; (TAT) n ...(A) protest; (ATIj n ...(AAT) n ; T C) n ...(T) n ; and (A) n (AAAC) n (A),
  • the SSR loci of the invention are preferably a maximum about 500 nucleotides long. In another preferred embodiment, the SSR locus of the invention is a minimum of
  • the invention further provides for isolated polynucleotides comprising at least one SSR motif and having the nucleotide sequences as shown in Table 3 (SEQ ID NOS: 237 to 354).
  • These polynucleotides may be of the same length as the sequences shown in Table 3 or alternatively comprise additional sequences on their 5', 3' or both ends.
  • the latter polynucleotides may be less than about 500bp, less than about lkb, less than about 2kb or less than about 3kb long.
  • the polynucleotides comprising the sequences of SEQ ID NOS: 237-354 do not containing any functional gene or coding sequences.
  • polynucleotides that (i) hybridize under the conditions of low, medium or high stringency to the polynucleotides comprising the sequences of SEQ ID NOS: 237-354 and (ii) contain SSR motifs.
  • these hybridizable polynucleotides are less than about lOOObp long, more preferably less than about 500bp long and most preferably less than about 200 bp long.
  • the hybridizable polynucleotide is about the same length as the polynucleotide to which it hybridizes.
  • polynucleotides that contain SSR motifs and have at least about 75%, preferably at least about 85%, and most preferably at least about 95% identity to the polynucleotides having the sequence of SEQ ID NOS:237 to 354.
  • Polynucleotides that contain SSR motifs and have at least about 75%, preferably at least about 85%, and most preferably at least about 95% homology to the polynucleotides having the sequence of SEQ ID NOS:237 to 354 are also within the scope of the invention.
  • polynucleotides that align to polynucleotides of SEQ LD NO:237-354 under the following conditions are also within the scope of the invention: alignment done using PowerBlast network client on
  • genomic DNA used is a plant DNA, more preferably the pine DNA and most preferably the DNA from the Pinus genus.
  • genomic DNA may be from P. taeda, P. caribaea, P. ponderosa, P. radiata, P. resinosa, P. strobus, or P. sylvestris. In one embodyment of the invention, these polynucleotides are less than about 500bp long.
  • the length of the amplified DNA fragment is generally limited only by the resolving power of the particular separation system used.
  • the thin denaturing gels for example, are capable of resolving fragments differing by as little as 1 base up to a total fragment length of about 300 bp.
  • Use of longer gels and longer electrophoresis times can extend the resolving power up to about 600 bp or more.
  • the longer the fragment the lower the proportion of its length is occupied by the SSR sequences, and hence the resolution is more difficult.
  • Oligonucleotide primer adapted for detection of SSR marker are also within the scope of the invention.
  • a suitable primer comprises at least the sequence of SEQ ID NOS: 1-236 and 367-390.
  • the present invention also provides probes specific to at least part of the aforesaid SSRs for delecting SSR markers using methods other than polymerase chain reaction, such as for example hybridization with labeled probes.
  • the probes useful in the invention may be any sequence comprising at least the sequence of SEQ ID NOS: 1-236, as well as any other probe that a person of skill in the art can construct based on the information of
  • the SSR loci of the invention may be polymo ⁇ hic. They may have a PIC of at least 309c (0.3); ⁇ reier ⁇ n> ⁇ _. ... a ⁇ least " • • • . (0.7); and mosl prefernhiy of al least 90% (0.9).
  • the polynucleotides and primers of the invention may be subcloned and introduced into various host cells according to methods well known in the art. The resulting clones and host cell are also within the scope of the invention. A person of skill in the art can make all such constructs and host cells using methods known in the art. However, the following non-limiting examples are provided below.
  • a large number of vectors including bacterial, fungal and plant vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts.
  • Non-limiting examples include pKK plasmids (Clonetech, Palo Alto, CA), pUC plasmids, pET plasmids (Novagen, Inc., Madison, WI), or pRSET or pREP (Invitrogen, San Diego, CA), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art.
  • Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes.
  • Suitable host cells may be transformed/transfected/infected as appropriate by any suitable method including electroporation, CaCl 2 mediated DNA uptake, fungal infection, microinjection, microprojectile transformation, or other established methods.
  • Appropriate host cells include bacteria, archaebacteria, fungi, especially yeast, and plant and animal cells. Of particular interest are E. coli, B. subtilis, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Schizosaccharomyces pombi, SF9 cells, C129 cells, 293 cells, Neurospora, CHO cells, COS cells, HeLa cells, and immortalized mammalian myeloid and lymphoid cell lines.
  • Preferred replication systems include M13, ColEl, SV40, baculovirus, lambda, adenovirus, and the like.
  • the present invention is also directed to a kit for the rapid analysis of one or more specific DNA polymo ⁇ hisms of the type described in this application.
  • the kit includes oligodeoxynucleotide primers for the amplification of fragments containing one or more SSR sequences.
  • the present invention provides for the methods of identifying and isolating SSR loci and iheir use as genetic markers.
  • a method for the identification from genomic DNA of a fragment comprising a SSR locus comprising the steps of: (i) contacting a DNA library with at least one hybridisation probe so as to identify a population of DNA fragments enriched for simple tandem repeats; (ii) isolating and cloning said population; and (iii) screening of the resulting DNA library so as to identify an individual fragment comprising a simple tandem repeat locus.
  • the DNA library may be a genomic DNA library; the genomic DNA library may be any convenient population of DNA fragments such as pine DNA, or subgenomic DNA libraries such as those generated by PCR from flow soiled chromosomes (see Telenius, H., et al., 1992, Genomics 3: 718-725).
  • the genomic DNA library may be obtained by restriction digestion of genomic DNA.
  • the average fragment size within the DNA library may be less than 1.5 kilobases and may be less than about one kilobase.
  • the fragment size may be from about 400 bp to about 1000 bp.
  • the hybridisation probe or set of probes may be immobilised on a solid phase such as a nylon membrane and may be used to identify a particular class of SSRs. Such classes may include dimeric, trimeric, tetrameric, pentameric and hexameric repeats.
  • Particular oligonucleotide probes for use in the present invention may include oligonucleotide probes comprising a repeated region of greater than 200 bp.
  • the probe may comprise repeats having at least 70%, such as 85% or 100%, identity to a given repeat sequence.
  • the hybridisation probe may be a set of probes comprising mixed trimeric or tetrameric repeat DNA or any other combination of various SSR motifs.
  • the population of DNA fragments enriched for SSR may be amplified prior to cloning and this may be effected by PCR amplification.
  • Universal linker sequences may be ligated to the ends of individual fragments, possibly prior to the enrichment procedure, and linker sequence specific primers may then be used to amplify the enriched population. Linker sequences may then be removed, for example by restriction digestion, prior to cloning.
  • a method for the identification from genomic DNA of a fragment comprising a SSR locus comprises the steps of: (i) ligating universal linker sequences to the ends of fragments comprised in a genomic DNA library so as to form a library for PCR amplification; (ii) contacting said PCR library with at least one hybridisation probe so as lo identify a population of library fragments enriched for simple tandem repeats; (iii) separating and amplifying said population by PCR; and (iv) cloning and screening the resulting amplification products so as to isolate an individual fragment comprising a simple tandem repeat locus.
  • Cloning may be effected using any convenient cloning procedure and vector (for example pBluescriptll (Stratagene, LaJolla, CA)) such as those described by Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989), Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press.
  • pBluescriptll Stratagene, LaJolla, CA
  • Screening may be effected using any convenient hybridisation probe or set of probes comprising SSR sequences. These may be the same as those disclosed above in respect of the enrichment procedure.
  • Individual clones comprising SSR loci may be analyzed using conventional techniques to determine, for example, specific sequence information. Such techniques may allow the generation of individual "identities" specific for one or more polymo ⁇ hic loci. The generation of such individuals "identities” may be used to identify and characterise family relationships and may be used for e.g. trait tracing in a breeding program and in any other technique which uses SSRs and their polymo ⁇ hisms. According to a further aspect of the present invention there are also provided methods of genetic characterisation wherein sample DNA is characterised by reference to at least one of the SSR loci, primer sequences and probes of the invention.
  • the method of genetic characterisation may comprise either the use of at least one hybridisation probe or it may comprise the use of polymerase chain reaction (PCR) primers specific to at least one of the SSR loci in order to amplify selectively the SSR locus.
  • the PCR primers may comprise at least one of the primers and probes of the present invention.
  • the method of genetic characterisation may be used in genetic mapping studies such as linkage studies, and may be used in the genetic analysis of inherited traits.
  • the present invention is directed to a method for detecting the presence of a specific trait in a subject, such as a plant.
  • the method includes isolating the genomic DNA from the subject individual and analyzing the genomic DNA with a polymo ⁇ hic amplified DNA marker containing one or more SSR sequences.
  • the analysis comprises amplification u.>mg the polymerase chain reaction of one or more short DNA fragments containing the SSR followed by measurement of the sizes of the amplified fragments using gel electrophoresis.
  • SSR markers of the invention for detection of polymo ⁇ hism in various pines are provided in the Examples. Any other known uses of such markers will be apparent to persons of skill in the art.
  • the source of DNA used for clone library construction was needle tissue from a vegetative clone of the P. taeda tree known as 7-56. Allele diversity surveys were based on needle tissue DNA from vegetative clones of 26 trees that were selected from breeding populations established from five geographic origins. The sample origins included South and North Carolina Atlantic Coastal Plain (15 trees) and Piedmont (2 trees), central
  • Taq DNA polymerase contained DNA fragments that provided non-specific polymerase priming sues during the primer extension step, thus primer exiension reaction components were treated with exonuclease I and lambda exonuclease to remove extraneous oligonucleotides and increase the proportion of SSR-specific extensions.
  • Alkaline phosphatase-conjugated oligonucleotide probes specific for each SSR motif were used for chemiluminescent detection and identification of bacterial clones carrying SSR inserts. Probe hybridizations were done on either colony lift, or 96-well arrayed, nylon membranes. Only a single round of SSR clone identification and isolation was used prior to DNA sequence analysis. Di- and trinucleotide primers and probes were all 30 nucleotides in length, while the tetranucleotide primers and probes were 32 nucleotides long. SSR-enriched libraries constructed using a different SSR enrichment strategy were obtained from Genetic Information Services, Inc. (Chatsworth, CA).
  • genomic DNA was partially digested with a cocktail of blunt-end-generating restriction endonucleases, and size fractionated and purified by agarose gel electrophoresis.
  • the purified fragments ranging from 350 to 650 bp. were ligated with adapter oligonucleotides to provide common PCR priming sites for all fragments, and to provide a H dIII restriction endonuclease site for subsequent cloning into a pUC19 plasmid vector.
  • the adapted fragments were denatured and hybridized to SSR oligonucleotides bound to magnetic microbeads. Non-SSR bearing fragments were washed away from the beads.
  • SSR bearing fragments were released by denaturation, PCR amplified, and used for clone library construction.
  • SSR probe hybridizations and detection to identify the SSR-bearing bacterial clones were done on 96-well arrayed nylon membranes, as described above.
  • Plasmid DNA for sequencing was prepared according to manufacturers' instructions using either Wizard Miniprep columns (Promega Co ⁇ ., Madison, WI) or
  • PCR amplification and agarose gel electrophoresis were done as described by Echt et al. (1996).
  • PCR amplification success and locus polymo ⁇ hism were evaluated on high-resolution agarose gels containing 3% TreviGel-500 (Trevigen, Inc., Gaithersburg, MD).
  • Precise allele sizing and locus genotyping were done by fluorescent marker analysis on an ABI373A Automated DNA Analyzer running GeneScan software (PE Applied Biosystems, Foster City, CA).
  • Polymo ⁇ hism potential in P. taeda was evaluated for each marker using one of two methods. In the first, 18 or 20 loblolly pine trees were genotyped for each marker (RIPPT1 through RIPPT89).
  • H heterozygosity
  • PIC polymo ⁇ hism information content
  • the PIC is the probability that the parental origin of an allele can be determined from the marker genotype of the locus in any given offspring (Botstein et al. 1980 Am. J. Hum. Genet., 32: 314-331.). Following Liu (1998, Statistical Genomics: Linkage, Mapping, and QTL Analysis, CRC Press, Boca Raton, Florida. 611 p.):
  • / is the number of codominant alleles at the locus
  • p is the frequency of homozygous genotypes
  • p is the frequency of ith ory ' th allele.
  • the second method used a two-step strategy to identify polymorphism.
  • For the primary screen an individual tree and a pool of eight other individuals from both ACP and non-ACP geographic origins were PCR amplified for each marker locus (RIPPTIOO through RIPPT9325), giving four samples of template DNA. Polymorphism was recorded when, following high resolution agarose gel electrophoresis, a pooled sample displayed more alleles than the individual sample from the same pool, or when size differences were observed between the ACP and non-ACP samples.
  • For the secondary screen single, polymo ⁇ hic SSR loci identified from the primary screen were amplified from eight individuals (four trees each from both the ACP and non-ACP groups), and allelic size differences were scored from high-resolution agarose gels.
  • the proportion of SSR clones in each of the enriched libraries varied from 1% to 15%, depending on the target motif, specific sequence of the oligonucleotide extension primer, and experimental conditions. No correlations were found between the SSR motif and the level of enrichment of a particular library. A total of 644 SSR clones were sequenced, with an average insert size of 400 bp.
  • Table 1 Frequency of sequenced cloned inserts, unique cloned sequences, primers pairs synthesized, single loci that were PCR amplified, and polymorphic SSR loci, by motif.
  • the trinucleotide repeats that were examined did not, in general, prove to be a very good source of polymo ⁇ hic markers despite their relative abundance in the pine genome reported by Echt and May-Marquardt 1997.
  • the three trinucleotide motifs that did produce polymo ⁇ hic markers had a sequence-to-marker conversion frequency of about 3.7% (Table 1).
  • the ATC motif the most abundant trinucleotide SSR in loblolly pine (Echt and May-Marquardt 1997), accounted for relatively few informative markers. It appeared to be associated with a repetitive DNA fraction in the genome, as only 36% of the (ATC) n clones sequenced were unique sequences.
  • RIPPTl GCATGCCAAAAGATCTCAA (SEQ ID NO 1 ) AGTGAACTCGGGAGGCTTCT (SEQ ID NO 2) IP 263 N RIPPT6 TTTGGACAAGTGGCTTGTTG (SEQ ID NO 3) ATGTTTGATTGCATGGGGAT (SEQ ID NO 4) IP 295 N RIPPTl 1 GGCTTCTCTCCAAGCTTTTTG (SEQ ID NO 5) GAATGAGCCTCCCAACTCAA (SEQ ID NO 6) IP 171 N RIPPT22 C ⁇ CAGTTTCATAATCTTTGTCGC (SEQ ID NO 7) TTTTAGAAAAGAAGGAAATCTTCA (SEQ ID NO 8) IP 250 N RIPPT24 GACACCGGATACTGAGGTGG (SEQ ID NO 9) CCCGCAACTTCGTAAGAGTC (SEQ ID NO 10) IP 151 N
  • RIPPT104 TGCATTTCATTTTTTTGCGTGT (SEQ ID NO 41) AGGACATGGAGAGTTTACACATG (SEQ ID NO 42) 1 164 N RIPPTl 06 ATCAGATTGGTGGATCGGAG (SEQ ID NO 43) TGACTGATAAGGGTTTCGCC (SEQ ID NO 44) 2 180 N RIPPTl 17 GCTTCATGATTTCTCGATCG (SEQ ID NO 45) TCTGCGTGGATAAAGGAATTT (SEQ ID NO 46) 2 208 N RIPPTl 23 TCGTGTCGAAACATTGGAAA (SEQ ID NO 47) TATCACCTATAGCCCCGTCG (SEQ ID NO 48) 1 ! 29 N
  • RIPPTl 26 TCATACCGAGAGAGGTCTTTG (SEQ ID NO 49) GAGCTTAATTTGTGCCTGCC (SEQ ID NO 50) 1 174 N RIPPTl 28 CGACCCTAGTCTCTTGTGCA (SEQ ID NO 51) TTTTGGACCCTAAGCCAGAG (SEQ ID NO 52) 1 175 Y RIPPTl 32 AACCGTGGTGCTCTGATACC (SEQ ID NO 53) TGCAAGTCAAGAGCTAGAGACAA (SEQ ID NO 54) 1 113 N RIPPTl 34 GTTTACATTTTCCTGGGGCA (SEQ ID NO 55) GATTTACAAAAATCCCTGCCA (SEQ ID NO 56) 1 145 N RIPPTl 35 CACGCATGAGCTGAGTCATAA (SEQ ID NO 57) TGTGTTTCCCACTATGCTAAGC (SEQ ID NO 58) 1 218 N
  • RIPPTl 39 ACCAACCGAGGGAGCTAAAT (SEQ ID NO 59) AAAAACGACATTCACTTCAACA (SEQ ID NO 60) 1 121 N RIPPT158 GTGTGCCACGGATGTATGAG (SEQ ID NO 61) TTGCTGAAAGGGCCAGTAGT (SEQ ID NO 62) 2 211 N RIPPT159 ATATGGCTTACCTCGGGTCC (SEQ ID NO 63) CATAAACCCATTGGGTCCAG (SEQ ID NO 64) 2 131 N RIPPT165 TGGAAGCCACAATTTGTTGA (SEQ ID NO 65) TGCAATAAAACCATGCAACAA (SEQ ID NO 66) 220 N RIPPT166 TTTTGAGAATGTCCGTGCG (SEQ ID NO 67) TGATGCATTGCAAAATCATG (SEQ ID NO 68) 155 Y
  • RIPPTl 71 TGATCCTAAGCCTTAGAAACCC (SEQ ID NO 69) TTTTGTCACCCATGCATATGA (SEQ ID NO 70) 207 N RIPPT179 TGTAGGAGCACAAGCCATTG (SEQ ID NO 71) AACACAGTTGGACCGTTTGA (SEQ ID NO 72) 170 N RIPPTl 85 TGTTTGCAAATCATGGGGTA (SEQ ID NO 73) CCAGTGTCCATGCCAATTTT (SEQ ID NO 74) 300 N RIPPT193 GATCCCTTGTCCCAGAAACA (SEQ ID NO 75) TGTTGATGTTATGCCTGGGT (SEQ ID NO 76) 163 N RIPPT211 GAGGGGGTCTCATACACCAA (SEQ ID NO 77) TGCATAGAGGATGTATTTCTTGGA (SEQ ID NO 78) 159 N
  • RIPPT388 CACAACACTCAAACATGCTCAA (SEQ ID NO 99) AAGAGGATGTGAGGTCCCAA (SEQ ID NO 100) 203 N RIPPT467 CTTGGCGACCTTGTCATACA (SEQ ID NO 101) GGGTCCTTAGGGATCATGGT (SEQ ID NO 102) 178 N RIPPT496 GTAAGAGTGCCTCGGGTCTG (SEQ ID NO 103) GGTGGTAGGTAGATCGGCAA (SEQ ID NO 104) 203 N
  • RIPPT508 GGCACAGGTTGGACATCTCT (SEQ ID NO: 105) GTGGTGGAAGGGAGATTTCA (SEQ ID NO: 106) 90 N RIPPT538 AAACACTTGGACTGGATGGG (SEQ ID NO: 107) TTTGGAGGATGTTTGTTGCA (SEQ ID NO: 108) 212 N RIPPT540 TGTTGTCATTAGTGGTAGGATCA (SEQ ID NO: 109) AAGCGATGTCACTTGTTGAGAA (SEQ ID NO: 1 10) 200 N RIPPT548 TTTTGTGGTCATTCGTTGGA (SEQ ID NO:l l l) TCACATGGAAGATTATCTCCAAA (SEQ ID NO: 112) 207 N RIPPT556 TCGTGATTACATTGCTGCCT (SEQ ID NO: 113) TCCACAACAATGATCGCTTC (SEQ ID NO:l 14) 183 N
  • RIPPT560 CATTGGAACTTCACCGAAGG (SEQ ID NO: 115) GTGCTATTGGGTCCAGCAAT (SEQ ID NO: 116) 108 N RIPPT567 GTTGGTGAGGAGACTTGGGA (SEQ ID NO: 117) AAGAACAATTCCA ATATGGATGA (SEQ ID NO: 118) 152 N R1PPT584 GCGAGACAGAAACGGAAAAG (SEQ ID NO: 119) CTCTGCTAGACCGCTCAGCT (SEQ ID NO: 120) 136 N RIPPT609 CAAAATGCAGAGGGGCTTAA (SEQ ID NO:121) CCAGTCCATCGAATCACGTA (SEQ ID NO: 122) 154 N RIPPT619 CAGCTCTCTTAATAGCCTCGG (SEQ ID NO:123) GCACATAGCAACGCTG A AGA (SEQ ID NO: 124) 191 N
  • RIPPT621 GCAAAGGGAAGCAAAGTCAT (SEQ ID NO: 125) TTCGTCCTCTTTTGAACGAGT (SEQ ID NO: 126) 154 N RIPPT627 GACAAACA ACCCTTGCGTTT (SEQ ID NO: 127) GACCCATCAAGCCAACATG (SEQ ID NO: 128) 168 N RIPPT629 GGTTGTGCTTTCCCAGAGAG (SEQ ID NO: 129) GAATGCAAGGTAGCCAGGAG (SEQ ID NO: 130) 157 N RIPPT630 CGCA AGCTATG ATACA ACGC (SEQ ID NO: 131 ) TGTTGGCTGAGTGTGAAAGC (SEQ ID NO: 132) 157 N RIPPT644 GTTGTGATCCAAGTCCCCTG (SEQ ID NO: 133) TGGTCCATTCGGTCCTATTC (SEQ ID NO: 134) 204 N
  • RIPPT688 TTCAGTTATGCATTCACGAGC (SEQ ID NO: 145) GTCCTCCTGGGTTATCCCTC (SEQ ID NO: 146) 141 N RIPPT689 GAAACTTTCCCCTACGAGCC (SEQ ID NO: 147) TTCCCCAAAAGTTCACAGGT (SEQ ID NO: 148) 158 N RIPPT690 ATTCCTAGATGGACCTGGGG (SEQ ID NO: 149) CGACATAAGCCCACCAAATT (SEQ ID NO: 150) 142 N RIPPT692 TGG ATCGTG ATCCTCTGTGA (SEQ ID NO: 151 ) GCTTCCATCACATTGGGATT (SEQ ID NO: 152) 166 Y RIPPT700 TTGCAATTGCGATTAACTGC (SEQ ID NO: 153) ATAATGGCATAGCCGAATCG (SEQ ID NO: 154) 180 N
  • RIPPT841 GTGCTTCCCTTGCTTCAGAC (SEQ ID NO: 171) GCAAATGCAAACTTTGGGTA (SEQ ID NO: 172) 1 202 N RIPPT846 CATTCATGGTTCCAATGTGG (SEQ ID NO: 173) TGATAAGCGTGGATCTCGTG (SEQ ID NO: 174) 2 109 N R1PPT852 OTTATCCCCCATGTTGTTGC (SEQ ID NO: 175) GGGTAGAAGCACTATGCTTTCATT (SEQ ID NO: 176) 2 213 N RIPPT860 TTGAGCAGACATCATCAACACT (SEQ ID NO: 177) CCAGGTTATGCCTCAAAGAG (SEQ ID NO: 178) 1 217 N RIPPT905 CACGGATCTCTGGAAACCAT (SEQ ID NO:1 9) CGCTGGTTTCCCTCAGAATA (SEQ ID NO: 180) 1 194 N
  • RIPPT921 GGATTTTGTTTTCCTCATAATCA (SEQ ID NO:181) GGGCATAGCATATGCCACTT (SEQ ID NO: 182) 1 219 Y RIPPT932 GCAAGACCGACTGGATTAGC (SEQ ID NO: 183) GAGGTCATGATATGTGGTGGG (SEQ ID NO: 184) 2 130 N RIPPT941 CTGCGTAGCAAATCACTGGA (SEQ ID NO: 185) TGATCTGATGTGGGATCAACA (SEQ ID NO: 186) 1 151 N RIPPT947 CCATTGCCCGAGCTAGTTTA (SEQ ID NO: 187) TTATATTGGACCCAAGGCCC (SEQ ID NO: 188) 1 214 N RIPPT958 TGGAGTCTCGAACACTGTGG (SEQ ID NO: 189) AATCATCCCAATGGCAACAT (SEQ ID NO: 190) 1 111 Y
  • RIPPT960 GCATCCATCTTCAGCATCCT (SEQ ID NO: 191 ) TTCATACGACACCTTTGAAATG (SEQ ID NO: 192) 1 188 N RIPPT961 CCATTAG AC A AGTGCGCATG (SEQ ID NO: 193) TGAAAAAGGAATTTCCCCAA (SEQ ID NO: 194) 1 213 N RIPPT968 TCTACGACAAAACCACGTAGTG (SEQ ID NO: 195) CATGTGGCTTTGTGGCATAT (SEQ ID NO: 196) 1 201 N RIPPT984 TGTGACCTGAAAATTCCCCT (SEQ ID NO: 197) GGCTTGCAACCAGTTCCATA (SEQ ID NO: 198) 1 220 N RIPPT990 GACCTAAAGAGGTTCACGCG (SEQ ID NO: 199) TCAAATCTTGGGTTAGTATGCAGA (SEQ ID NO: 200) 1 220 N
  • RIPPT1037 TGCTCAATATAGACCACTTGCA (SEQ ID NO:211) AGCCATAATTCAACAAAAGGAA (SEQ ID NO:212) 1 152 N RIPPTl 040 TCAAGGAATTCATTGGAGCC (SEQ ID NO:213) TTTGGCCATATCAAACCCAT (SEQ ID NO:214) 1 192 N RIPPT1066 AAAGGGGGTGTTTGATGGAT (SEQ ID NO:215) GATCGAAATCAGCGAACACA (SEQ ID NO:216) 1 175 Y
  • RIPPTl 072 TTTCATGACCTTGGAGTGGA (SEQ ID NO:217) ATTGATCCCATTGTTGCTCC (SEQ ID NO:218) 1 209 N RIPPTl 076 TGTGTAAACCCAGGCTAGGC (SEQ ID NO:219) ATGATTTCACAAAGCCCCTC (SEQ ID NO:220) 1 167 N RIPPT1077 AACATTCTAGCATGCCCCAC (SEQ ID NO:221) TTGTGGTGGATGTCTCCT (SEQ ID NO:222) 1 220 N RIPPTl 125 GAGCCACACAAACATGCATC (SEQ ID NO:223) TTTCCCAAAAGTTCACGAGG (SEQ ID NO: 224) 2 197 Y RIPPTl 137 CCCATGCAACTGCCTAGAAT (SEQ ID NO:225) AAGCTCGCACGTGGGATA (SEQ ID NO:226) 2 165 N RIPPT9058 CCCGCTCCTATTCAAGATCA (SEQ ID NO:22
  • Polymo ⁇ hism among P. taeda individuals was scored using high resolution agarose gel electrophoresis. Heterozygous marker alleles having a 3 bp size difference could be resolved and 2 bp allele size differences between samples in adjacent lanes could be detected. Since single bp allelic differences were not detectable some 2 bp allelic differences were possibly missed, the number of polymo ⁇ hic loci reported may be slightly underestimated. In table 1, the number of polymo ⁇ hic single loci reflects the number of primer pairs generating a single major DNA fragment, or a heterozygote fragment pattern. For most primer pairs, there was only one fragment amplified.
  • SSR loci of P. taeda identified using the primer pairs in Table 2 are represented in Table 3.
  • RIPPTL LOBSEQ3-27-97ATC441R (SEQ. ID. NO. 237) ATTAATTTTTTTTGAAAAAAAAAGAGTTTTGAGAAAAAGTCTAATATATACTTGGTGGCATGCCAAAAGATCTCAAAAA
  • TTCCT TCATATATTTGATTAGACAAGAAAATATATTATATTATAATCGTTTAAC ⁇ TTT ⁇ ATAATTTTAAAAAATATATTA TAATTATTTTAAGTTTATGATGATGATGATGGTGGTTAACGTCCACTGAGACCAAATAATGATCATCGGACCT AAAAAGACAAATTATTTATTTTGATTTAAGATRTTATTCCTATGCTCAAAAAGCRTGCAGAGAAGCCTCCCGAGTTCACT ATAATTTTGGCATTGTAAAAGGNTAGGAAAGGTCATTGGTGGTTACAAAGGGTGGTGAAATTGAAATCTAATGTTGGTG TTTGCCGGGGCTTCC
  • RIPPT22 LOBSEQ5-2-97ATC272 (SEQ. ID. NO. 240) ACAACCACATTAGATCTCAGTTTCATAATCTTTGTCGCAATACTGACCTTCCTAGCCTTTTACGATGTCATAATTATAGT
  • RIPPT31 LOBSEQAAT18 (SEQ. ID. NO. 242) ATTGTTC ⁇ TCTGGATTAATTACACTAGTAATTT ⁇ TCAAATCAAAGTTTCAAACCAACCAATGTGGTTCATCATCAAAATA TAGATGAGGGAGGTTGAACTAAGCCATCGAGATTGATAAGAGGACTGGCAATCTGAACATAGATAATGGGTGGAAAAT
  • RIPPT66 LOBSEQ6-5-97AAATE2 (SEQ. ID. NO. 247) ATTATTTTTATGTAGGCTTTGATTATATTGGTTCCCCTTAGACTCCTATATATAGAAAGGAGGTCTTGTCATTTGTATCAT
  • RIPPT69 LOBSEQ6-5-97AAATE9 (SEQ. ID. NO. 249) ATCAAGAATGGGGGATGATTCACCATTTTTGGAGTAAAAGGATAAAAATAAATAAATAAAAATAAAACCTTATT
  • RIPPT79 AAT5 (SEQ. ID. NO. 252) ACTTTATATAGCATTTAAAAACACAATTTAAATGATGAAAAGTCACACATTGTATATTTAAAAAGACATAAGCACCCTA
  • RIPPT101 pPT101.seq (SEQ. ID. NO.255) ATC ⁇ GGTAAT ⁇ C GTTATGATCATGATTATGC ⁇ GATGTTTATATATGTATACATGTAGGTGTGTATGTATGTCATGTTTGA
  • RIPPT126 pPT126.seq (SEQ. ID. NO. 261) CC ⁇ CAANGCTAAGANGC ⁇ TTACTGTAAATCATACCGAGAGAGGTCTTTGTAAAAATCATGTGTGTGTGTGTGTGTGTG
  • TAGTCATGGATACACT TATGTTCCTTATAATGTGGTAAATATAACTTATAAGTGTGAATGCATTAGCGACGAACCCACC TAATATTAATAGCACRAAGGGAACCACGCTATAATTGTTTGGATTAATATTTGGTTGTCATATTATAATATTGGGANGTG ACC ⁇ ACCI AAAATGTTTCTCGAAGGGCTCTTTGGTCTCTAGCAATCATACAAAGANG
  • RIPPTl 35 pPT135.seq (SEQ. ID. NO. 265) ATCTTTTCAATARRTAACATTGAAAAGCATTAAAGAATAGCATTTTGACAACTAAGGGTGAATACCCAAATTCATACAC
  • RIPPT158 pPT158.seq (SEQ. ID. NO. 267) ACTAGAGGCACACAGTGGGAGTCTCAGATCGGATCCACCGACTACTTAGTAATGTTGCACGAGTTGTCTCGTGCTACCA
  • RIPPT165 pPT165.seq (SEQ. ID. NO. 269) CCTTGTGGAAGCCACAATTTGTTGAGTATTGGCAATTATTGAAAAAACCCirrCAAGCTCTTGAATCTGTATTCGTCCTC ⁇ GAACGAGTCT , CTCT , c ⁇ crc ⁇ c ⁇ c ⁇ c ⁇ c ⁇ c ⁇ c ⁇ c ⁇ c ⁇ 'crrc ⁇ c ⁇ c ⁇ ACACACATATTCATGGGTATGTTCAACTTTGATGTGTTTGTTTTTTGTTC
  • RIPPT179 pPT179.seq (SEQ. ID. NO. 272) ATCATTTATTTCAAAACATGTAAAAAAAATAAACATGTAGGAGCACAAGCCATTGATTATTTTCTCTATTTTTTAAAGGA
  • RIPPT305 pPT305.seq (SEQ. ID. NO. 282) TCAATCACCAATTATTTGGCrNTCTAGGTGTTTTTTTTCATATACCTAGATCGAGTCT NGCCI ⁇
  • CTrCATCTTCATCTAATTTC CCCCTTCrTTAGACTTrGTrATATGTGGCATAGTTTCATCCACTCCGAC
  • RIPPT367 pPT367.seq (SEQ. ID. NO. 283) CCrCTATTTGAAATGTGATCATCATATTGGACTTATAAGAGGTAACATATAACATACATTTCCAAAACTTTCGTAAGGA GATCAACACTCTCCAAATAATTAGGAATCCCTCCTTCTAAGGTCAATCATAGGCATAAACCATGGATATAAATACAT
  • RIPPT560 pPT560.seq (SEQ. ID. NO. 294) ATCGCAATATAGCATTGGAACTTCACCGAAGGGCGAAGCTATACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACA
  • RIPPT990 pPT990.seq (SEQ. ID. NO. 336) ATCACTGGAAAGCTCTTAATGAGCTAAACACGATGGTAATTTTTTTTTAAAGTI TGATGAGTTTGGAAAAAAGATGATT
  • ATCACCTAGTCTGCCCCTAGTGTGATGTTTCI ATCTCCAAAGAGTCTTCCTTGTAAACGAGACTCACAAAGTGAATTTT TTCACTC ⁇ TTATTTTAC ⁇ AATTTGAAGTTTTCATGACCTTGGAGTGGATTCACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACA
  • TTG 4 (TC) 2 (TTA) I2 (SEQ ID NO:356) 3 146-152 0.282 0.320
  • Loci RIPPTl, RIPPT6, RIPPT32, RIPPT64, RIPPT65, RIPPT71, and RIPPT80 had minimum size differences among some alleles of a single base pair.
  • the lbp allele size differences may have originated in short, mutable mononucleotide stretches found near or adjacent to the target SSR and included in the PCR amplified region.
  • the RIPPT71 locus had no repeats other than (AAAT) n . SSR markers in other pines
  • Table 5 includes individual species results for RIPPTl through RIPPT90, while Table 6 is a summary of success of amplification of single loci patterns in other species for all RIPPT primer pairs.
  • polymo ⁇ hism among species was scored from high resolution agarose gels, so the number of polymo ⁇ hic SSR loci amplified among species may have been underestimated. Details of the agarose gel marker phenotypes are given only for the 49 primer pairs that amplified single loci in P. taeda among RIPPTl through RIPPT90 (Table 5).
  • SSR marker phenotypes among various pine species using primer pairs that amplified single loci in P. taeda.
  • (+) indicateds that one or more fragments were amplified outside of the expected size range, which is ⁇ 100 bp from the marker size in P. taeda. 1 integers indicate the number of PCR fragments observed in the expected size range.
  • P. P. P. P. P. P. P. P. caribaea ponderosa radiata resinosa strobus sylvestris
  • GGTCCCATAGACCAATTTGG (SEQ ID NO:367) RIPPT7 GATCAATCATCAAATTCATCACC (SEQ ID NO:368) (CAT) 6 113
  • TCATGGGGTCAATTCTCCTC SEQ ID NO:375
  • RIPPT30 ATGGATGGAAAATTTCTATAGCC (SEQ ID NO:376) (ATT), 3 236
  • ATGTTTCCAATTAAAGGATTTCC (SEQ ID NO:377) RIPPT58 GCCTTGCAAAGTGACCTCTC (SEQ ID NO:378) (AGG) 4 240

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Botany (AREA)
  • Mycology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention porte sur des marqueurs microsatellites, à répétition de séquence (SSR), qui possèdent un fort potentiel pour améliorer la cartographie génomique et la recherche d'identification de génotype dans la génétique forestière et l'arboriculture. Les marqueurs SSR ont été développés par isolement et séquençage des clones SSR du pin négresse 1539 pour les motifs structuraux SSR 11. Après avoir recherché les redondances dans les séquences, 566 paires d'amorces oligonucléotidiques de la PCR sur le flanc des SRR ont été synthétisées et évaluées pour leur capacité à amplifier l'ADN génomique à partir du pin négresse. Les trois motifs structuraux des SSR qui ont produit la plus importante proportion de marqueurs informatifs à partir des clones séquencés étaient (AC)n, (AAAT)n, et (AAAC)n. Dix-huit loci SSR polymorphes tri- et té tranucléotidiques ont été génotypés dans 20 pins négresse par analyse automatique des marqueurs fluorescents. Le nombre moyen des allèles par locus observé était de 6,4, et le contenu moyen des informations sur le polymorphisme (PIC) était de 0,547. Les sous-ensembles des 566 amorces primaires ont été évalués pour leur capacité à amplifier l'ADN à partir de six espèces d'autres pins, et 54 marqueurs amplifiés par les paires d'amorces qui étaient des polymorphes parmi les espèces. Cette invention porte également sur des procédés d'utilisation des loci SSR identifiés comme marqueurs génétiques.
PCT/US2000/000325 1999-01-15 2000-01-06 Marqueurs d'adn microsatellites et leurs utilisations WO2000042210A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU24076/00A AU2407600A (en) 1999-01-15 2000-01-06 Microsatellite dna markers and uses thereof
NZ512941A NZ512941A (en) 1999-01-15 2000-01-06 Length polymorphisms in microsatellite (simple sequence repeat - SSR) DNA markers and their use as genetic markers in pine

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US23288499A 1999-01-15 1999-01-15
US09/232,884 1999-01-15
US09/232,785 1999-01-19
US09/232,785 US6733965B2 (en) 1999-01-15 1999-01-19 Microsatellite DNA markers and uses thereof

Publications (2)

Publication Number Publication Date
WO2000042210A2 true WO2000042210A2 (fr) 2000-07-20
WO2000042210A3 WO2000042210A3 (fr) 2007-06-14

Family

ID=26926330

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/000325 WO2000042210A2 (fr) 1999-01-15 2000-01-06 Marqueurs d'adn microsatellites et leurs utilisations

Country Status (3)

Country Link
AU (1) AU2407600A (fr)
NZ (1) NZ512941A (fr)
WO (1) WO2000042210A2 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005085288A2 (fr) * 2004-03-01 2005-09-15 The Cbr Institute For Biomedical Research Anticorps naturels igm et leurs inhibiteurs
EP1591536A1 (fr) * 2004-04-29 2005-11-02 Institut National de la Recherche Agronomique Procédé pour la sélection des génotypes tournesol à haute teneur en acide oléique dans l'huile des graines
ES2351326A1 (es) * 2009-06-12 2011-02-03 Instituto Nacional De Investigaciones Y Tecnologia Agraria Y Alimentaria (Inia) Metodo para la identificacion del origen de los piñones comerciales.
CN104278097A (zh) * 2014-09-30 2015-01-14 中国科学院武汉植物园 用于猕猴桃杂交群体雌雄性别鉴定的ssr分子标记a003
CN104293942A (zh) * 2014-09-30 2015-01-21 中国科学院武汉植物园 用于猕猴桃杂交群体雌雄性别鉴定的ssr分子标记a001
CN104313019A (zh) * 2014-09-30 2015-01-28 中国科学院武汉植物园 用于猕猴桃杂交群体雌雄性别鉴定的ssr分子标记a002
AU2015202476B2 (en) * 2004-04-06 2017-06-22 Fibria Celulose S/A Cambium/xylem-preferred promoters and uses thereof
CN107557362A (zh) * 2017-10-26 2018-01-09 南京林业大学 一种马尾松cpSSR多态性引物及其松树近缘种的鉴定方法
CN108676907A (zh) * 2018-06-04 2018-10-19 贵州师范大学 一种基于转录组测序获得青篱柴ssr引物的方法
CN115323060A (zh) * 2021-06-11 2022-11-11 中国水产科学研究院珠江水产研究所 一种鱼类核基因分子标记引物、分子标记及分子标记数据库
CN116179738A (zh) * 2022-08-31 2023-05-30 中国医学科学院药用植物研究所海南分所 一种用于鉴定沉香品种的ssr分子标记的核心引物组及应用
CN116200521A (zh) * 2022-12-05 2023-06-02 东北林业大学 一种鉴别红松无性系的ssr标记引物组及指纹图谱的构建方法和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5378602A (en) * 1991-05-29 1995-01-03 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Highly informative microsatellite repeat polymorphic DNA markers twenty-[seven]six
US5721100A (en) * 1991-05-29 1998-02-24 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Three highly informative microsatellite repeat polymorphic DNA markers
US5843647A (en) * 1993-12-21 1998-12-01 University Of Leicester Simple tandem repeats

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5378602A (en) * 1991-05-29 1995-01-03 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Highly informative microsatellite repeat polymorphic DNA markers twenty-[seven]six
US5721100A (en) * 1991-05-29 1998-02-24 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Three highly informative microsatellite repeat polymorphic DNA markers
US5843647A (en) * 1993-12-21 1998-12-01 University Of Leicester Simple tandem repeats

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005085288A2 (fr) * 2004-03-01 2005-09-15 The Cbr Institute For Biomedical Research Anticorps naturels igm et leurs inhibiteurs
WO2005085288A3 (fr) * 2004-03-01 2006-08-03 Cbr Inst For Biomedical Res Anticorps naturels igm et leurs inhibiteurs
EP1725659A2 (fr) * 2004-03-01 2006-11-29 The CBR Institute for Biomedical Research, Inc. Anticorps naturels igm et leurs inhibiteurs
US7442783B2 (en) 2004-03-01 2008-10-28 The Cbr Institute For Biomedical Research, Inc. Natural IgM antibodies and inhibitors thereof
US9914751B2 (en) 2004-03-01 2018-03-13 Children's Medical Center Corporation Natural IGM antibodies and inhibitors thereof
US9657060B2 (en) 2004-03-01 2017-05-23 Children's Medical Center Corporation Natural IgM antibodies and inhibitors thereof
AU2015202476B2 (en) * 2004-04-06 2017-06-22 Fibria Celulose S/A Cambium/xylem-preferred promoters and uses thereof
EP1591536A1 (fr) * 2004-04-29 2005-11-02 Institut National de la Recherche Agronomique Procédé pour la sélection des génotypes tournesol à haute teneur en acide oléique dans l'huile des graines
ES2351326A1 (es) * 2009-06-12 2011-02-03 Instituto Nacional De Investigaciones Y Tecnologia Agraria Y Alimentaria (Inia) Metodo para la identificacion del origen de los piñones comerciales.
CN104293942B (zh) * 2014-09-30 2016-04-20 中国科学院武汉植物园 用于猕猴桃杂交群体雌雄性别鉴定的ssr分子标记a001
CN104278097A (zh) * 2014-09-30 2015-01-14 中国科学院武汉植物园 用于猕猴桃杂交群体雌雄性别鉴定的ssr分子标记a003
CN104313019A (zh) * 2014-09-30 2015-01-28 中国科学院武汉植物园 用于猕猴桃杂交群体雌雄性别鉴定的ssr分子标记a002
CN104293942A (zh) * 2014-09-30 2015-01-21 中国科学院武汉植物园 用于猕猴桃杂交群体雌雄性别鉴定的ssr分子标记a001
CN104278097B (zh) * 2014-09-30 2016-01-06 中国科学院武汉植物园 用于猕猴桃杂交群体雌雄性别鉴定的ssr分子标记a003
CN107557362B (zh) * 2017-10-26 2019-08-23 南京林业大学 一种马尾松cpSSR多态性引物及其松树近缘种的鉴定方法
CN107557362A (zh) * 2017-10-26 2018-01-09 南京林业大学 一种马尾松cpSSR多态性引物及其松树近缘种的鉴定方法
CN108676907A (zh) * 2018-06-04 2018-10-19 贵州师范大学 一种基于转录组测序获得青篱柴ssr引物的方法
CN115323060A (zh) * 2021-06-11 2022-11-11 中国水产科学研究院珠江水产研究所 一种鱼类核基因分子标记引物、分子标记及分子标记数据库
CN115323060B (zh) * 2021-06-11 2023-08-01 中国水产科学研究院珠江水产研究所 一种鱼类核基因分子标记引物、分子标记及分子标记数据库
CN116179738A (zh) * 2022-08-31 2023-05-30 中国医学科学院药用植物研究所海南分所 一种用于鉴定沉香品种的ssr分子标记的核心引物组及应用
CN116179738B (zh) * 2022-08-31 2023-09-22 中国医学科学院药用植物研究所海南分所 一种用于鉴定沉香品种的ssr分子标记的核心引物组及应用
CN116200521A (zh) * 2022-12-05 2023-06-02 东北林业大学 一种鉴别红松无性系的ssr标记引物组及指纹图谱的构建方法和应用
CN116200521B (zh) * 2022-12-05 2023-08-18 东北林业大学 一种鉴别红松无性系的ssr标记引物组及指纹图谱的构建方法和应用

Also Published As

Publication number Publication date
AU2407600A (en) 2000-08-01
NZ512941A (en) 2003-09-26
WO2000042210A3 (fr) 2007-06-14

Similar Documents

Publication Publication Date Title
US20030049612A1 (en) Microsatelite dna markers and uses thereof
Joshi et al. Molecular markers in plant genome analysis
Groppe et al. A microsatellite marker for studying the ecology and diversity of fungal endophytes (Epichloë spp.) in grasses
Rowland et al. Development of EST-PCR markers for DNA fingerprinting and genetic relationship studies in blueberry (Vaccinium, section Cyanococcus)
RU2748688C2 (ru) Генетический локус, ассоциированный с гнилью корня и стебля, обусловленной phytophthora, у сои
US20100021916A1 (en) Microsatellite-based fingerprinting system for saccharum complex
WO2000042210A2 (fr) Marqueurs d'adn microsatellites et leurs utilisations
Kim et al. Development of cultivar-specific DNA markers based on retrotransposon-based insertional polymorphism in Japanese pear
MX2014000608A (es) Marcadores de ssr para plantas y usos de los mismos.
WO2003085133A2 (fr) Nouvelles amorces fissr-pcr et methode d'identification de genotypage pour divers genomes de systemes vegetaux et animaux, dont des varietes de riz, et trousse correspondante
US5874217A (en) Microsatellite sequences for canine genotyping
Bekkaoui et al. Application of for the identification and management of hybrid poplar accessions
Akhare et al. RAPD profile studies in Sorghum for identification of hybrids and their parents
EP1546177A2 (fr) Empreinte genetique pour le cannabis sativa (marijuana) au moyen de marqueurs microsatellites (str)
US10590491B2 (en) Molecular markers associated with Mal de Rio Cuarto Virus in maize
US20240090396A1 (en) Clubroot resistance in brassica
JP4366494B2 (ja) アブラナ科植物根こぶ病に対する抵抗性遺伝子検出用マイクロサテライトマーカー、およびその利用
Echt et al. Microsatellite DNA markers and uses thereof
Triwitayakorn et al. Analysis of genetic diversity of the Thai swamp buffalo (Bubalus bubalis) using cattle microsatellite DNA markers
EP0857219B1 (fr) Sequences microsatellites destinees a la determination du genotype de races canines
US20130212734A1 (en) Genetic loci associated with resistance of corn to fijivirus
CN114717350B (zh) 水稻株型的分子标记及其应用
US10883148B2 (en) Molecular markers associated with Orobanche resistance in sunflower
Palaniappan et al. RAPD and ISSR analyses reveal low levels of genetic diversity in Phyllanthus amarus
AU777197B2 (en) Molecular markers in ryegrasses and fescues

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 512941

Country of ref document: NZ

122 Ep: pct application non-entry in european phase